Many mutations resulting in neurodevelopmental and neuropsychiatric disorders target synaptic proteins. Synapse remodeling and loss precedes cell death in neurodegenerative diseases, such as Alzheimer's or Parkinson's, and addictive drugs are known to alter synaptic function. The convergence of many brain disorders at the synapse, indicate that its integrity is critical to normal brain function. Monitoring synapse lifetime and assembly/disassembly in vivo has been hampered by the difficulty of discretely labeling and simultaneously tracking the recruitment and assembly of its individual components. Technology for robust, real-time visualization of synapse formation and loss in vivo would enable the exploration of this fundamental feature of brain development and plasticity, and its dysfunction in brain disease. Our goal is to address this need by developing high-resolution, high throughput temporal focusing (TF) two-photon microscopy for large-volume imaging of synapse assembly-disassembly in the living mouse brain. We propose two aims: 1) Design and implement TF two-photon microscopy for imaging an entire neocortical neuron at synaptic resolution in vivo in less than one minute. Imaging small structures in vivo, especially within the context of the full dendritic arbor, imposes significant time demands due to the need for increased sampling and longer dwell times. Currently, imaging an entire neuron at synaptic resolution takes 60-90 minutes. It is impossible to track events on the order of hours or minutes with such long scan times, or to stably image in awake mice. We propose a novel parallelized approach, line scanning TF two-photon microscopy, to enable in vivo imaging with throughput at least two orders of magnitude higher than point scanning, but with comparable resolution and signal-to-noise ratio. We will test the feasibility of this approach for imaging synaptic structural dynamics in real time, in the awake mouse. 2) Incorporate multi-spectral capabilities into a TF imaging system to enable in vivo tracking of multiple synaptic labels across a single neuron. Visualizing multiple discrete subcellular structures in vivo requires methods for efficient spectrally resolved imaging in deep tissue. We have achieved simultaneous three-color imaging with a single focus scanning multiphoton microscope using Ti-Sapphire lasers and an optical parametric amplifier (OPA) as light sources. However, these devices do not provide light pulses with sufficient peak power for highly parallelized imaging. We will extend the capability of the high-throughput line-scan TF imaging system by implementing multi-color excitation using a regenerative amplifier delivering femtosecond pulses at 1040 nm combined with a tunable OPA providing additional pulses in the 650-1600 nm range. This will allow simultaneous excitation of a broad palette of fluorescent proteins, enabling the tracking of multiple synaptic components at once. The technology we propose will provide a new and powerful tool for dissecting the synaptic roots of many disorders that affect formation, stability, and plasticity of excitatory and inhibitory synapses.